Activity-based protein profiling and global proteome analysis reveal MASTL as a potential therapeutic target in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a prevalent malignancy with limited therapeutic options for advanced stages. This study aimed to identify novel therapeutic targets for GC by profiling HSP90 client kinases. METHODS: We used mass spectrometry-based activity-based protein profiling (ABPP) with a desthiobiotin-ATP probe, combined with sensitivity analysis of HSP90 inhibitors, to profile kinases in a panel of GC cell lines. We identified kinases regulated by HSP90 in inhibitor-sensitive cells and investigated the impact of MASTL knockdown on GC cell behavior. Global proteomic analysis following MASTL knockdown was performed, and bioinformatics tools were used to analyze the resulting data. RESULTS: Four kinases-MASTL, STK11, CHEK1, and MET-were identified as HSP90-regulated in HSP90 inhibitor-sensitive cells. Among these, microtubule-associated serine/threonine kinase-like (MASTL) was upregulated in GC and associated with poor prognosis. MASTL knockdown decreased migration, invasion, and proliferation of GC cells. Global proteomic profiling following MASTL knockdown revealed NEDD4-1 as a potential downstream mediator of MASTL in GC progression. NEDD4-1 was also upregulated in GC and associated with poor prognosis. Similar to MASTL inhibition, NEDD4-1 knockdown suppressed migration, invasion, and proliferation of GC cells. CONCLUSIONS: Our multi-proteomic analyses suggest that targeting MASTL could be a promising therapy for advanced gastric cancer, potentially through the reduction of tumor-promoting proteins including NEDD4-1. This study enhances our understanding of kinase signaling pathways in GC and provides new insights for potential treatment strategies.

NAD+ Precursors Reverse Experimental Diabetic Neuropathy in Mice.

Abnormal NAD+ signaling has been implicated in axonal degeneration in diabetic peripheral neuropathy (DPN). We hypothesized that supplementing NAD+ precursors could alleviate DPN symptoms through increasing the NAD+ levels and activating the sirtuin-1 (SIRT1) protein. To test this, we exposed cultured Dorsal Root Ganglion neurons (DRGs) to Nicotinamide Riboside (NR) or Nicotinamide Mononucleotide (NMN), which increased the levels of NAD+, the SIRT1 protein, and the deacetylation activity that is associated with increased neurite growth. A SIRT1 inhibitor blocked the neurite growth induced via NR or NMN. We then induced neuropathy in C57BL6 mice with streptozotocin (STZ) or a high fat diet (HFD) and administered NR or NMN for two months. Both the STZ and HFD mice developed neuropathy, which was reversed through the NR or NMN administration: sensory function improved, nerve conduction velocities normalized, and intraepidermal nerve fibers were restored. The NAD+ levels and SIRT1 activity were reduced in the DRGs from diabetic mice but were preserved with the NR or NMN treatment. We also tested the effect of NR or NMN administration in mice that overexpress the SIRT1 protein in neurons (nSIRT1 OE) and found no additional benefit from the addition of the drug. These findings suggest that supplementing with NAD+ precursors or activating SIRT1 may be a promising treatment for DPN.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration.

BACKGROUND: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin-proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. RESULTS: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. CONCLUSIONS: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy.

Axon degeneration in diabetic peripheral neuropathy (DPN) is associated with impaired NAD+ metabolism. We tested whether the administration of NAD+ precursors, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), prevents DPN in models of Type 1 and Type 2 diabetes. NMN was administered to streptozotocin (STZ)-induced diabetic rats and STZ-induced diabetic mice by intraperitoneal injection at 50 or 100 mg/kg on alternate days for 2 months. mice The were fed with a high fat diet (HFD) for 2 months with or without added NR at 150 or 300 mg/kg for 2 months. The administration of NMN to STZ-induced diabetic rats or mice or dietary addition of NR to HFD-fed mice improved sensory function, normalized sciatic and tail nerve conduction velocities, and prevented loss of intraepidermal nerve fibers in skin samples from the hind-paw. In adult dorsal root ganglion (DRG) neurons isolated from HFD-fed mice, there was a decrease in NAD+ levels and mitochondrial maximum reserve capacity. These impairments were normalized in isolated DRG neurons from NR-treated mice. The results indicate that the correction of NAD+ depletion in DRG may be sufficient to prevent DPN but does not significantly affect glucose tolerance, insulin levels, or insulin resistance.

LAPTM4α is targeted from the Golgi to late endosomes/lysosomes in a manner dependent on the E3 ubiquitin ligase Nedd4-1 and ESCRT proteins.

Lysosome-associated protein transmembrane 4α (LAPTM4α) is a four transmembrane-spanning protein primarily localized in endosomes and lysosomes and has several putative lysosomal targeting signals at its C-terminal cytoplasmic domain, including tyrosine-based motifs (YxxΦ) and PY motifs (L/PxxY). LAPTM4α has been previously shown to be ubiquitinated by the E3 ubiquitin ligase Nedd4-1 through binding to its PY motifs and sorted to lysosomes, however, the molecular mechanisms underlying the localization of LAPTM4α to endosomes/lysosomes have not yet been fully elucidated. In the present study, we show that LAPTM4α binds Nedd4-1 in a manner dependent on PY motifs, while the PY motifs and Nedd4-1 are not necessarily required for LAPTM4α ubiquitination. The binding of LAPTM4α with Nedd4-1, however, is necessary for an effective sorting of LAPTM4α from the Golgi to late endosomes/lysosomes. An unexpected finding is that LAPTM4α is localized in the lumen, but not in the limiting membrane, of late endosomes, and degraded in lysosomes over time. Interestingly, we further found that siRNA knockdown of endosomal sorting complexes required for transport (ESCRT) components that mediate sorting of ubiquitinated membrane proteins into intralumenal vesicles (ILVs) of endosomes selectively blocks the transport of LAPTM4α to endosomes. Collectively, these results suggest that trafficking of LAPTM4α from the Golgi to endosomes is promoted by the interaction with Nedd4-1, which further requires ESCRT components. Furthermore, our findings highlight a novel function for ESCRT proteins in mediating protein and/or vesicle trafficking from the Golgi to endosomes/lysosomes.

The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2-HO-1 Axis.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. METHODS: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. RESULTS: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. CONCLUSIONS: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.

Sonic hedgehog signaling instigates high-fat diet-induced insulin resistance by targeting PPARγ stability.

Obesity is a major risk for patients with chronic metabolic disorders including type 2 diabetes. Sonic hedgehog (Shh) is a morphogen that regulates the pancreas and adipose tissue formation during embryonic development. Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily and one of the most important regulators of insulin action. Here, we evaluated the role and mechanism of Shh signaling in obesity-associated insulin resistance and characterized its effect on PPARγ. We showed that Shh expression was up-regulated in subcutaneous fat from obese mice. In differentiated 3T3-L1 and primary cultured adipocytes from rats, recombinant Shh protein and SAG (an agonist of Shh signaling) activated an extracellular signal-regulated kinase (ERK)-dependent noncanonical pathway and induced PPARγ phosphorylation at serine 112, which decreased PPARγ activity. Meanwhile, Shh signaling degraded PPARγ protein via binding of PPARγ to neural precursor cell-expressed developmentally down-regulated protein 4-1 (NEDD4-1). Furthermore, vismodegib, an inhibitor of Shh signaling, attenuated ERK phosphorylation induced by a high fat diet (HFD) and restored PPARγ protein level, thus ameliorating glucose intolerance and insulin resistance in obese mice. Our finding suggests that Shh in subcutaneous fat decreases PPARγ activity and stability via activation of an ERK-dependent noncanonical pathway, resulting in impaired insulin action. Inhibition of Shh may serve as a potential therapeutic approach to treat obesity-related diabetes.

The NEDD4-1 E3 ubiquitin ligase: A potential molecular target for bortezomib sensitivity in multiple myeloma.

E3 ubiquitin ligases primarily determine the substrate specificity of the ubiquitin-proteasome system and play an essential role in the resistance to bortezomib in multiple myeloma (MM). Neural precursor cell-expressed developmentally downregulated gene 4-1 (NEDD4-1, also known as NEDD4) is a founding member of the NEDD4 family of E3 ligases and is involved in the proliferation, migration, invasion and drug sensitivity of cancer cells. In the present study, we investigated the role of NEDD4-1 in MM cells and explored its underlying mechanism. Clinically, low NEDD4-1 expression has been linked to poor prognosis in patients with MM. Functionally, NEDD4-1 knockdown (KD) resulted in bortezomib resistance in MM cells in vitro and in vivo. The overexpression (OE) of NEDD4-1, but not an enzyme-dead NEDD4-1-C867S mutant, had the opposite effect. Furthermore, the overexpression of NEDD4-1 in NEDD4-1 KD cells resensitized the cells to bortezomib in an add-back rescue experiment. Mechanistically, pAkt-Ser473 levels and Akt signaling were elevated and decreased by NEDD4-1 KD and OE, respectively. NEDD4-1 ubiquitinated Akt and targeted pAkt-Ser473 for proteasomal degradation. More importantly, the NEDD4-1 KD-induced upregulation of Akt expression sensitized MM cells to growth inhibition after treatment with an Akt inhibitor. Collectively, our results suggest that high NEDD4-1 levels may be a potential new therapeutic target in MM.

Nicotinamide Mononucleotide Administration Prevents Experimental Diabetes-Induced Cognitive Impairment and Loss of Hippocampal Neurons.

Diabetes predisposes to cognitive decline leading to dementia and is associated with decreased brain NAD+ levels. This has triggered an intense interest in boosting nicotinamide adenine dinucleotide (NAD+) levels to prevent dementia. We tested if the administration of the precursor of NAD+, nicotinamide mononucleotide (NMN), can prevent diabetes-induced memory deficits. Diabetes was induced in Sprague-Dawley rats by the administration of streptozotocin (STZ). After 3 months of diabetes, hippocampal NAD+ levels were decreased (p = 0.011). In vivo localized high-resolution proton magnetic resonance spectroscopy (MRS) of the hippocampus showed an increase in the levels of glucose (p < 0.001), glutamate (p < 0.001), gamma aminobutyric acid (p = 0.018), myo-inositol (p = 0.018), and taurine (p < 0.001) and decreased levels of N-acetyl aspartate (p = 0.002) and glutathione (p < 0.001). There was a significant decrease in hippocampal CA1 neuronal volume (p < 0.001) and neuronal number (p < 0.001) in the Diabetic rats. Diabetic rats showed hippocampal related memory deficits. Intraperitoneal NMN (100 mg/kg) was given after induction and confirmation of diabetes and was provided on alternate days for 3 months. NMN increased brain NAD+ levels, normalized the levels of glutamate, taurine, N-acetyl aspartate (NAA), and glutathione. NMN-treatment prevented the loss of CA1 neurons and rescued the memory deficits despite having no significant effect on hyperglycemic or lipidemic control. In hippocampal protein extracts from Diabetic rats, SIRT1 and PGC-1α protein levels were decreased, and acetylation of proteins increased. NMN treatment prevented the diabetes-induced decrease in both SIRT1 and PGC-1α and promoted deacetylation of proteins. Our results indicate that NMN increased brain NAD+, activated the SIRT1 pathway, preserved mitochondrial oxidative phosphorylation (OXPHOS) function, prevented neuronal loss, and preserved cognition in Diabetic rats.

Distinguishing the optimal binding mechanism of an E3 ubiquitin ligase: Covalent versus noncovalent inhibition.

The development of covalent drugs, specifically in cancer therapeutics, has recently sparked interest among the pharmaceutical research community. While representing a significant fraction of the drugs in the market, very few have been deliberately designed to interact covalently with their biological target. One of the enzymes that have been both covalently and noncovalently targeted is the Neural Precursor Cell Expressed Developmentally Downregulated gene 4-1 (Nedd4-1). This enzyme has been found to have multiple physiological implications, including its involvement in cancer invasion. A critical gap still remains in the molecular understanding of the structural mechanism upon the covalent and noncovalent binding to Nedd4-1. In this study, we explore the most optimal binding mechanism in the inhibition of the catalytic site of the Nedd4-1. Our results exhibited a greater stability in the covalent complex compared with the noncovalent complex. This was supported by the secondary structure elements that were more dominant in the covalently inhibited complex. This complex disclosed an optimal free binding energy landscape, induced by the catalytic site energy contributions that showed to be more favorable. The insights demonstrating the above binding mechanism of Nedd4-1 establishes covalent inhibition as the preferred method of inhibition of the enzyme. This investigation aids in the understanding of the structural mechanism of Nedd4-1 inhibition and would assist in the design of more potent covalent inhibitors at the catalytic site of Nedd4-1.

Evidence that melatonin downregulates Nedd4-1 E3 ligase and its role in cellular survival.

Indolamine melatonin structurally resembles non-covalent proteasome inhibitors; however, the role of ubiquitin proteasome system (UPS) in neuronal survival and how melatonin carries out UPS inhibition remain largely unknown. With the use of melatonin treated cells, we evaluated the expression of Nedd4-1, an E3 ligase, how melatonin regulates its activity and its relationship with neuronal survival. Nedd4-1 was upregulated in the hypoxic condition in both control and Nedd4-1 overexpressed cells and melatonin treatment reversed its expression in both normoxic and hypoxic conditions, which was associated with increased cellular survival. Melatonin had no effect on the expression of Nedd4-1 at mRNA level. However, when melatonin was administered along with protein synthesis inhibitor cycloheximide, protein level of Nedd4-1 was further reduced, indicating that melatonin possibly downregulates Nedd4-1 after its synthesis. Notably, co-immunoprecipitation analyses followed by Liquid chromatography-Mass Spectrometry (LC-MS/MS) revealed that melatonin may dissociate ribosomal proteins, such as RS19, RL23A, and nucleophosmin from Nedd4-1, while 40S ribosomal protein S7 and 60S ribosomal protein L35 came into contact with Nedd4-1 upon melatonin treatment. By using IPA analyses, we obtained further data indicated novel target molecules of melatonin in hypoxic conditions, including OTOF, SF3B2, IPO5, ST13, FGFR3, Mx1/Mx2, playing roles in RNA splicing and trafficking, growth factor and interferon signaling. Here, we described a new insight into the role of melatonin in UPS functioning by proposing a molecular mechanism through which melatonin regulates Nedd4-1.

Promotion of liver growth by CAR is accompanied by Akt pathway activation and FoxM1-Nedd4-mediated repression of PTEN.

Recently, we reported that treatment with the mouse agonist of the constitutive androstane receptor (CAR), 1,4-bis benzene[2-(3,5-dichloropyridyloxy)] (TCPOBOP; a well-known hepatomitogen), reduced PTEN protein levels, leading to Akt activation. Hence, the present study was performed to demonstrate the role of CAR in PTEN regulation and liver growth. Liver hyperplasia caused by CAR activation was confirmed to be mediated by a decrease in PTEN protein level and the activation of the Akt signalling pathway in the liver of mice. Treatment with the CAR agonist decreased the PTEN levels and increased Foxm1 levels, which correlate with the elevated expression of the FoxM1 target gene, Nedd4-1, an E3 ligase involved in PTEN ubiquitination, and the promotion of degradation. The increase in Nedd4-1 levels was accompanied by an increase in CAR-mediated accumulation of Foxm1 on the Nedd4-1 gene promoter. Therefore, these results provide evidence that a notable function of CAR is its liver growth promotion effect, which is accompanied by FoxM1-Nedd4-mediated repression of PTEN and Akt pathway activation.

Molecular mechanisms of Streptococcus pneumoniae-targeted autophagy via pneumolysin, Golgi-resident Rab41, and Nedd4-1-mediated K63-linked ubiquitination.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

The Adaptor Protein Alix is Involved in the Interaction Between the Ubiquitin Ligase NEDD4-1 and its Targets, ABCG1 and ABCG4.

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer's disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.

Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase through Target-Focused Modelling.

The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the enzyme may be targeted both covalently and non-covalently, minimal studies provide effective inhibitors against it. Recently, research has focused on covalent inhibitors based on their characteristic, highly-selective warheads and ability to prevent drug resistance. This prompted us to screen for new covalent inhibitors of Nedd4-1 using a combination of computational approaches. However, this task proved challenging due to the limited number of electrophilic moieties available in virtual libraries. Therefore, we opted to divide an existing covalent Nedd4-1 inhibitor into two parts: a non-covalent binding group and a pre-selected α, ß-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on molecular interactions at the binding site. The pharmacophore was then subjected to virtual screening to identify structurally similar hit compounds. Multiple filtrations were implemented prior to selecting four hits, which were validated with a covalent conjugation and later assessed by molecular dynamic simulations. The results showed that, of the four hit molecules, Zinc00937975 exhibited advantageous molecular groups, allowing for favourable interactions with one of the characteristic cysteine residues. Predictive pharmacokinetic analysis further justified the compound as a potential lead molecule, prompting its recommendation for confirmatory biological evaluation. Our inhouse, refined, pharmacophore model approach serves as a robust method that will encourage screening for novel covalent inhibitors in drug discovery.

Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron.

Kir4.1/5.1 heterotetramer participates in generating the negative cell membrane potential in distal convoluted tubule (DCT) and plays a critical role in determining the activity of Na-Cl cotransporter (NCC). Kir5.1 contains a phosphothreonine motif at its COOH terminus (AA249-252). Coimmunoprecipitation showed that Nedd4-2 was associated with Kir5.1 in HEK293 cells cotransfected with Kir5.1 or Kir4.1/Kir5.1. GST pull-down further confirmed the association between Nedd4-2 and Kir5.1. Ubiquitination assay showed that Nedd4-2 increased the ubiquitination of Kir4.1/Kir5.1 heterotetramer in the cells cotransfected with Kir4.1/Kir5.1, but it has no effect on Kir4.1 or Kir5.1 alone. Patch-clamp and Western blot also demonstrated that coexpression of Nedd4-2 but not Nedd4-1 decreased K currents and Kir4.1 expression in the cells cotransfected with Kir4.1 and Kir5.1. In contrast, Nedd4-2 fails to inhibit Kir4.1 in the absence of Kir5.1 or in the cells transfected with the inactivated form of Nedd4-2 (Nedd4-2C821A). Moreover, the mutation of TPVT motif in the COOH terminus of Kir5.1 largely abolished the association of Nedd4-2 with Kir5.1 and abolished the inhibitory effect of Nedd4-2 on K currents in HEK293 cells transfected with Kir4.1 and Kir5.1 mutant (Kir5.1T249A). Finally, the basolateral K conductance in the DCT and Kir4.1 expression is significantly increased in the kidney-specific Nedd4-2 knockout or in Kir5.1 knockout mice in comparison to their corresponding wild-type littermates. We conclude that Nedd4-2 binds to Kir5.1 at the phosphothreonine motif of the COOH terminus, and the association of Nedd4-2 with Kir5.1 facilitates the ubiquitination of Kir4.1, thereby regulating its plasma expression in the DCT.

Clinicopathological and prognostic correlations of HER3 expression and its degradation regulators, NEDD4-1 and NRDP1, in primary breast cancer.

BACKGROUND: Human epidermal growth factor receptor HER3 (ErbB3), especially in association with its relative HER2 (ErbB2), is known as a key oncogene in breast tumour biology. Nonetheless, the prognostic relevance of HER3 remains controversial. NEDD4-1 and NRDP1 are signalling molecules closely related to the degradation of HER3 via ubiquitination. NEDD4-1 and NRDP1 have been reported to contribute to HER3-mediated signalling by regulating its localization and cell membrane retention. We studied correlations between HER3, NEDD4-1, and NRDP1 protein expression and their association with tumour histopathological characteristics and clinical outcomes. METHODS: The prevalence of immunohistochemically detectable expression profiles of HER3 (n = 177), NEDD4-1 (n = 145), and NRDP1 (n = 145) proteins was studied in primary breast carcinomas on archival formalin-fixed paraffin-embedded (FFPE) samples. Clinicopathological correlations were determined statistically using Pearson's Chi-Square test. The Kaplan-Meier method, log-rank test (Mantel-Cox), and Cox regression analysis were utilized for survival analysis. RESULTS: HER3 protein was expressed in breast carcinomas without association with HER2 gene amplification status. Absence or low HER3 expression correlated with clinically aggressive features, such as triple-negative breast cancer (TNBC) phenotype, basal cell origin (cytokeratin 5/14 expression combined with ER negativity), large tumour size, and positive lymph node status. Low total HER3 expression was prognostic for shorter recurrence-free survival time in HER2-amplified breast cancer (p = 0.004, p = 0.020 in univariate and multivariate analyses, respectively). The majority (82.8%) of breast cancers demonstrated NEDD4-1 protein expression - while only a minor proportion (8.3%) of carcinomas expressed NRDP1. NEDD4-1 and NRDP1 expression were not associated with clinical outcomes in HER2-amplified breast cancer, irrespective of adjuvant trastuzumab therapy. CONCLUSIONS: Low HER3 expression is suggested to be a valuable prognostic biomarker to predict recurrence in HER2-amplified breast cancer. Neither NEDD4-1 nor NRDP1 demonstrated relevance in prognostics or in the subclassification of HER2-amplified breast carcinomas.

Aß-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-ß (Aß). Specifically, it has been shown that Aß decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aß-induced synaptic dysfunction in cultured rat neurons. We find that Aß promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aß-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aß. SIGNIFICANCE STATEMENT: Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aß. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aß-mediated control of a ubiquitin ligase to regulate surface AMPAR expression.

Type I γ Phosphatidylinositol Phosphate 5-Kinase i5 Controls the Ubiquitination and Degradation of the Tumor Suppressor Mitogen-inducible Gene 6.

Mitogen-inducible gene 6 (Mig6) is a tumor suppressor, and the disruption of Mig6 expression is associated with cancer development. Mig6 directly interacts with epidermal growth factor receptor (EGFR) to suppress the activation and downstream signaling of EGFR. Therefore, loss of Mig6 enhances EGFR-mediated signaling and promotes EGFR-dependent carcinogenesis. The molecular mechanism modulating Mig6 expression in cancer remains unclear. Here we demonstrate that type I γ phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), stabilizes Mig6 expression. Knockdown of PIPKIγi5 leads to the loss of Mig6 expression, which dramatically enhances and prolongs EGFR-mediated cell signaling. Loss of PIPKIγi5 significantly promotes Mig6 protein degradation via proteasomes, but it does not affect the Mig6 mRNA level. PIPKIγi5 directly interacts with the E3 ubiquitin ligase neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1). The C-terminal domain of PIPKIγi5 and the WW1 and WW2 domains of NEDD4-1 are required for their interaction. The C2 domain of NEDD4-1 is required for its interaction with PtdIns(4,5)P2 By binding with NEDD4-1 and producing PtdIns(4,5)P2, PIPKIγi5 perturbs NEDD4-1-mediated Mig6 ubiquitination and the subsequent proteasomal degradation. Thus, loss of NEDD4-1 can rescue Mig6 expression in PIPKIγi5 knockdown cells. In this way, PIPKIγi5, NEDD4-1, and Mig6 form a novel molecular nexus that controls EGFR activation and downstream signaling.

NEDD4-1 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis via the PI3K/Akt pathway.

Activation of the Akt pathway has been shown to protect the heart from ischaemia/reperfusion (I/R) injury. NEDD4-1 has been shown to positively regulate nuclear trafficking of the activated form of Akt. However, the role of NEDD4-1 in cardiac I/R injury remains to be elucidated. In the present study, Lentiviral vectors were constructed to overexpress or knockdown NEDD4-1 in H9c2 cardiomyocytes subjected to I/R injury or ischemic preconditioning (IPC). The results indicated that NEDD4-1 levels were decreased after I/R and increased after IPC in rat heart tissue and in H9c2 cardiomyocytes. Overexpression of NEDD4-1 activated the Akt pathway and regulated apoptosis-related proteins in H9c2 cardiomyocytes, attenuating SI/R-induced cell apoptosis and caspase 3/7 activities. Furthermore, in vivo overexpression of NEDD4-1 attenuated myocardial apoptosis following myocardial I/R. Our results demonstrated that NEDD4-1 protects the myocardium from I/R induced apoptosis by activating PI3K/Akt signaling.